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DOE OSTI2017

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A 2A adenosine receptor (A 2A AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A 2A AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A 2A AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. Furthermore, these developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1990

A bit serial sequential circuit

Normally a sequential circuit with n state variables consists of n unique hardware realizations, one for each state variable. All variables are processed in parallel. This paper introduces a new sequential circuit architecture that allows the state variables to be realized in a serial manner using only one next state logic circuit. The action of processing the state variables in a serial manner has never been addressed before. This paper presents a general design procedure for circuit construction and initialization. Utilizing pass transistors to form the combinational next state forming logic in synchronous sequential machines, a bit serial state machine can be realized with a single NMOS pass transistor network connected to shift registers. The bit serial state machine occupies less area than other realizations which perform parallel operations. Moreover, the logical circuit of the bit serial state machine can be modified by simply changing the circuit input matrix to develop an adaptive state machine.

Hu, S.↗

NASA NTRS2005

Highly Reliable, High-Speed, Unidirectional Serial Data Links

Highly reliable, high-speed, unidirectional serial data-communication subsystems have been proposed to be installed in an upgrade of the computing systems aboard the space shuttles. The basic design concept of these serial data links is also adaptable to terrestrial use in applications in which there are requirements for highly reliable serial data communications. The hardware and software aspects of the architecture of the data links are dictated largely by a requirement, in the original space-shuttle application, for one computer to monitor the memory transactions and memory contents of other computers in real time with high reliability and without reliance on requests for retransmission. To minimize weight while affording a capability to transfer data at a required rate of 2.56 x 10(exp 8) bits per second, it was decided that the links would be serial ones of the fiber-channel type. [Fiber channel denotes a type of serial computer bus that is used to connect a computer (usually a supercomputer) with a high-speed data storage device. Depending on the specific application, the physical connection between the transmitter and receiver could be made via an optical fiber or a twisted pair of wires.] Heretofore, fiber-channel links have ordinarily been bidirectional and have operated under protocols that provide for receiving stations to detect errors and request retransmission when necessary. In the present case, the time taken by processing to request retransmission would conflict with the requirement for real-time transfer of data. To ensure reliability without retransmission, a link according to the proposal would utilize a modified version of the normal fiberchannel character set in conjunction with forward error correction by means of a Reed-Solomon code (see figure). The Reed-Solomon encoding and decoding and the translations between the normal and modified character sets would be effected by logic circuitry external to the fiber-channel transmitter and receiver, which would be commercial off-the-shelf units. The receiving end of the link could detect and correct errors at a rate as high as 4 million times per second, if necessary. The receiver detects uncorrectable double-byte errors. It has been estimated that uncorrectable-error rate would amount to one failure in about 10(exp 19) characters.

Cole, Robert M.↗

DOE OSTI2018

Solving protein structure from sparse serial microcrystal diffraction data at a storage-ring synchrotron source

In recent years, the success of serial femtosecond crystallography and the paucity of beamtime at X-ray free-electron lasers have motivated the development of serial microcrystallography experiments at storage-ring synchrotron sources. However, especially at storage-ring sources, if a crystal is too small it will have suffered significant radiation damage before diffracting a sufficient number of X-rays into Bragg peaks for peak-indexing software to determine the crystal orientation. As a consequence, the data frames of small crystals often cannot be indexed and are discarded. Introduced here is a method based on the expand–maximize–compress (EMC) algorithm to solve protein structures, specifically from data frames for which indexing methods fail because too few X-rays are diffracted into Bragg peaks. The method is demonstrated on a real serial microcrystallography data set whose signals are too weak to be indexed by conventional methods. In spite of the daunting background scatter from the sample-delivery medium, it was still possible to solve the protein structure at 2.1 Å resolution. The ability of the EMC algorithm to analyze weak data frames will help to reduce sample consumption. Furthermore, it will also allow serial microcrystallography to be performed with crystals that are otherwise too small to be feasibly analyzed at storage-ring sources.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

DOE OSTI2020

An automated platform for in situ serial crystallography at room temperature

Direct observation of functional motions in protein structures is highly desirable for understanding how these nanomachineries of life operate at the molecular level. Because cryogenic temperatures are non-physiological and may prohibit or even alter protein structural dynamics, it is necessary to develop robust X-ray diffraction methods that enable routine data collection at room temperature. We recently reported a crystal-on-crystal device to facilitate in situ diffraction of protein crystals at room temperature devoid of any sample manipulation. Here an automated serial crystallography platform based on this crystal-on-crystal technology is presented. A hardware and software prototype has been implemented, and protocols have been established that allow users to image, recognize and rank hundreds to thousands of protein crystals grown on a chip in optical scanning mode prior to serial introduction of these crystals to an X-ray beam in a programmable and high-throughput manner. This platform has been tested extensively using fragile protein crystals. We demonstrate that with affordable sample consumption, this in situ serial crystallography technology could give rise to room-temperature protein structures of higher resolution and superior map quality for those protein crystals that encounter difficulties during freezing. This serial data collection platform is compatible with both monochromatic oscillation and Laue methods for X-ray diffraction and presents a widely applicable approach for static and dynamic crystallographic studies at room temperature.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2017

Asymmetry in serial femtosecond crystallography data

Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2017

Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Plug-and-play polymer microfluidic chips for hydrated, room temperature, fixed-target serial crystallography

The practice of serial X-ray crystallography (SX) depends on efficient, continuous delivery of hydrated protein crystals while minimizing background scattering. Of the two major types of sample delivery devices, fixed-target devices offer several advantages over widely adopted jet injectors, including: lower sample consumption, clog-free delivery, and the ability to control on-chip crystal density to improve hit rates. In this study we present our development of versatile, inexpensive, and robust polymer microfluidic chips for routine and reliable room temperature serial measurements at both synchrotrons and X-ray free electron lasers (XFELs). Our design includes highly X-ray-transparent enclosing thin film layers tuned to minimize scatter background, adaptable sample flow layers tuned to match crystal size, and a large sample area compatible with both raster scanning and rotation based serial data collection. The optically transparent chips can be used both for in situ protein crystallization (to eliminate crystal handling) or crystal slurry loading, with prepared samples stable for weeks in a humidified environment and for several hours in ambient conditions. Serial oscillation crystallography, using a multi-crystal rotational data collection approach, at a microfocus synchrotron beamline (SSRL, beamline 12-1) was used to benchmark the performance of the chips. Furthermore, high-resolution structures (1.3–2.7 Å) were collected from five different proteins – hen egg white lysozyme, thaumatin, bovine liver catalase, concanavalin-A (type VI), and SARS-CoV-2 nonstructural protein NSP5. Overall, our modular fabrication approach enables precise control over the cross-section of materials in the X-ray beam path and facilitates chip adaption to different sample and beamline requirements for user-friendly, straightforward diffraction measurements at room temperature.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1999

Punctured Parallel and Serial Concatenated Convolutional Codes for BPSK/QPSK Channels

As available bandwidth for communication applications becomes scarce, bandwidth-efficient modulation and coding schemes become ever important. Since their discovery in 1993, turbo codes (parallel concatenated convolutional codes) have been the center of the attention in the coding community because of their bit error rate performance near the Shannon limit. Serial concatenated convolutional codes have also been shown to be as powerful as turbo codes. In this dissertation, we introduce algorithms for designing bandwidth-efficient rate r = k/(k + 1),k = 2, 3,..., 16, parallel and rate 3/4, 7/8, and 15/16 serial concatenated convolutional codes via puncturing for BPSK/QPSK (Binary Phase Shift Keying/Quadrature Phase Shift Keying) channels. Both parallel and serial concatenated convolutional codes have initially, steep bit error rate versus signal-to-noise ratio slope (called the -"cliff region"). However, this steep slope changes to a moderate slope with increasing signal-to-noise ratio, where the slope is characterized by the weight spectrum of the code. The region after the cliff region is called the "error rate floor" which dominates the behavior of these codes in moderate to high signal-to-noise ratios. Our goal is to design high rate parallel and serial concatenated convolutional codes while minimizing the error rate floor effect. The design algorithm includes an interleaver enhancement procedure and finds the polynomial sets (only for parallel concatenated convolutional codes) and the puncturing schemes that achieve the lowest bit error rate performance around the floor for the code rates of interest.

Acikel, Omer Fatih↗

NASA NTRS2005

Serial Back-Plane Technologies in Advanced Avionics Architectures

Current back plane technologies such as VME, and current personal computer back planes such as PCI, are shared bus systems that can exhibit nondeterministic latencies. This means a card can take control of the bus and use resources indefinitely affecting the ability of other cards in the back plane to acquire the bus. This provides a real hit on the reliability of the system. Additionally, these parallel busses only have bandwidths in the 100s of megahertz range and EMI and noise effects get worse the higher the bandwidth goes. To provide scalable, fault-tolerant, advanced computing systems, more applicable to today s connected computing environment and to better meet the needs of future requirements for advanced space instruments and vehicles, serial back-plane technologies should be implemented in advanced avionics architectures. Serial backplane technologies eliminate the problem of one card getting the bus and never relinquishing it, or one minor problem on the backplane bringing the whole system down. Being serial instead of parallel improves the reliability by reducing many of the signal integrity issues associated with parallel back planes and thus significantly improves reliability. The increased speeds associated with a serial backplane are an added bonus.

Varnavas, Kosta↗

NASA NTRS2003

Bit-Serial Adder Based on Quantum Dots

A proposed integrated circuit based on quantum-dot cellular automata (QCA) would function as a bit-serial adder. This circuit would serve as a prototype building block for demonstrating the feasibility of quantum-dots computing and for the further development of increasingly complex and increasingly capable quantum-dots computing circuits. QCA-based bit-serial adders would be especially useful in that they would enable the development of highly parallel and systolic processors for implementing fast Fourier, cosine, Hartley, and wavelet transforms. The proposed circuit would complement the QCA-based circuits described in "Implementing Permutation Matrices by Use of Quantum Dots" (NPO-20801), NASA Tech Briefs, Vol. 25, No. 10 (October 2001), page 42 and "Compact Interconnection Networks Based on Quantum Dots" (NPO-20855), which appears elsewhere in this issue. Those articles described the limitations of very-large-scale-integrated (VLSI) circuitry and the major potential advantage afforded by QCA. To recapitulate: In a VLSI circuit, signal paths that are required not to interact with each other must not cross in the same plane. In contrast, for reasons too complex to describe in the limited space available for this article, suitably designed and operated QCA-based signal paths that are required not to interact with each other can nevertheless be allowed to cross each other in the same plane without adverse effect. In principle, this characteristic could be exploited to design compact, coplanar, simple (relative to VLSI) QCA-based networks to implement complex, advanced interconnection schemes. To enable a meaningful description of the proposed bit-serial adder, it is necessary to further recapitulate the description of a quantum-dot cellular automation from the first-mentioned prior article: A quantum-dot cellular automaton contains four quantum dots positioned at the corners of a square cell. The cell contains two extra mobile electrons that can tunnel (in the quantum-mechanical sense) between neighboring dots within the cell. The Coulomb repulsion between the two electrons tends to make them occupy antipodal dots in the cell. For an isolated cell, there are two energetically equivalent arrangements (denoted polarization states) of the extra electrons. The cell polarization is used to encode binary information. Because the polarization of a nonisolated cell depends on Coulomb-repulsion interactions with neighboring cells, universal logic gates and binary wires could be constructed, in principle, by arraying QCA of suitable design in suitable patterns. Again, for reasons too complex to describe here, in order to ensure accuracy and timeliness of the output of a QCA array, it is necessary to resort to an adiabatic switching scheme in which the QCA array is divided into subarrays, each controlled by a different phase of a multiphase clock signal. In this scheme, each subarray is given time to perform its computation, then its state is frozen by raising its inter-dot potential barriers and its output is fed as the input to the successor subarray. The successor subarray is kept in an unpolarized state so it does not influence the calculation of preceding subarray. Such a clocking scheme is consistent with pipeline computation in the sense that each different subarray can perform a different part of an overall computation. In other words, QCA arrays are inherently suitable for pipeline and, moreover, systolic computations. This sequential or pipeline aspect of QCA would be utilized in the proposed bit-serial adders.

Fijany, Amir↗

NASA NTRS2012

High-Speed, Multi-Channel Serial ADC LVDS Interface for Xilinx Virtex-5 FPGA

Analog-to-digital converters (ADCs) are used in scientific and communications instruments on all spacecraft. As data rates get higher, and as the transition is made from parallel ADC designs to high-speed, serial, low-voltage differential signaling (LVDS) designs, the need will arise to interface these in field programmable gate arrays (FPGAs). As Xilinx has released the radiation-hardened version of the Virtex-5, this will likely be used in future missions. High-speed serial ADCs send data at very high rates. A de-serializer instantiated in the fabric of the FPGA could not keep up with these high data rates. The Virtex-5 contains primitives designed specifically for high-speed, source-synchronous de-serialization, but as supported by Xilinx, can only support bitwidths of 10. Supporting bit-widths of 12 or more requires the use of the primitives in an undocumented configuration, a non-trivial task. A new SystemVerilog design was written that is simpler and uses fewer hardware resources than the reference design described in Xilinx Application Note XAPP866. It has been shown to work in a Xilinx XC5VSX24OT connected to a MAXIM MAX1438 12-bit ADC using a 50-MHz sample clock. The design can be replicated in the FPGA for multiple ADCs (four instantiations were used for a total of 28 channels).

Taylor, Gregory H.↗

NASA NTRS2015

Fast Grasp Contact Computation for a Serial Robot

A system includes a controller and a serial robot having links that are interconnected by a joint, wherein the robot can grasp a three-dimensional (3D) object in response to a commanded grasp pose. The controller receives input information, including the commanded grasp pose, a first set of information describing the kinematics of the robot, and a second set of information describing the position of the object to be grasped. The controller also calculates, in a two-dimensional (2D) plane, a set of contact points between the serial robot and a surface of the 3D object needed for the serial robot to achieve the commanded grasp pose. A required joint angle is then calculated in the 2D plane between the pair of links using the set of contact points. A control action is then executed with respect to the motion of the serial robot using the required joint angle.

Shi, Jianying↗

DOE OSTI2021

Serial communication tapping and transmission to routable networks

Apparatuses and methods for tapping serial communications and transforming the serial data into a format appropriate for routable networks are significant for purposes of security and troubleshooting, especially in critical infrastructure networks. Communication taps should be completely passive such that any failure would not interrupt the serial communications. Furthermore, automatic determination of unspecified serial protocol frames allow general implementation across various networks, or across devices within a single network, without the need to customize for each implementation.

Edgar, Thomas W.↗

DOE OSTI2024

Serial communication tapping and transmission to routable networks

Edgar, Thomas W.↗

DOE OSTI2017

Acquisition of Real-Time Operation Analytics for an Automated Serial Sectioning System

Mechanical serial sectioning is a highly repetitive technique employed in metallography for the rendering of 3D reconstructions of microstructure. While alternate techniques such as ultrasonic detection, micro-computed tomography, and focused ion beam milling have progressed much in recent years, few alternatives provide equivalent opportunities for comparatively high resolutions over significantly sized cross-sectional areas and volumes. To that end, the introduction of automated serial sectioning systems has greatly heightened repeatability and increased data collection rates while diminishing opportunity for mishandling and other user-introduced errors. Unfortunately, even among current, state-of-the-art automated serial sectioning systems, challenges in data collection have not been fully eradicated. Therefore, this paper highlights two specific advances to assist in this area; a non-contact laser triangulation method for assessment of material removal rates and a newly developed graphical user interface providing real-time monitoring of experimental progress. Furthermore, both are shown to be helpful in the rapid identification of anomalies and interruptions, while also providing comparable and less error-prone measures of removal rate over the course of these long-term, challenging, and innately destructive characterization experiments.

36 MATERIALS SCIENCE↗

DOE OSTI2018

High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner

The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1 µm beam size and photon flux of 3 × 10 12 photons s −1 at a photon energy of 12.66 keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100 nm raster-scanning precision at over 10 grid-linepairs s −1 frequency for fly scans of a 200 µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750 Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.

43 PARTICLE ACCELERATORS↗

DOE OSTI2019

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, `naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗